During GABAergic synaptic transmission, G protein-coupled GABAB receptors (GBRs) activate K+ channels that prolong the duration of inhibitory postsynaptic potentials (IPSPs). We now show that KCTD16, an auxiliary GBR subunit, anchors hyperpolarization-activated cyclic nucleotide-gated (HCN) channels containing HCN2/HCN3 subunits to GBRs. In dopamine neurons of the VTA (DAVTA neurons), this interaction facilitates activation of HCN channels via hyperpolarization during IPSPs, counteracting the GBR-mediated late phase of these IPSPs. Consequently, disruption of the GBR/HCN complex in KCTD16-/- mice leads to prolonged optogenetic inhibition of DAVTA neuron firing. KCTD16-/- mice exhibit increased anxiety-like behavior in response to stress - a behavior replicated by CRISPR/Cas9-mediated KCTD16 ablation in DAVTA neurons or by intra-VTA infusion of an HCN antagonist in wild-type mice. Our findings support that the retention of HCN channels at GABAergic synapses by GBRs in DAVTA neurons provides a negative feedback mechanism that restricts IPSP duration and mitigates the development of anxiety.
- MeSH
- Dopaminergic Neurons * metabolism MeSH
- Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels * metabolism genetics MeSH
- Inhibitory Postsynaptic Potentials physiology drug effects MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Receptors, GABA-B * metabolism MeSH
- Ventral Tegmental Area * metabolism MeSH
- Anxiety * metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
N-Methyl-d-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type (WT) Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons from L825V/+ compared with +/+ mice. The peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSCs) was unchanged, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared with +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared with WT GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD, and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.
- MeSH
- Excitatory Postsynaptic Potentials physiology MeSH
- HEK293 Cells MeSH
- Hippocampus metabolism MeSH
- Humans MeSH
- Mutation, Missense MeSH
- Mice, Inbred C57BL MeSH
- Mice, Transgenic * MeSH
- Mice MeSH
- Neurons metabolism MeSH
- Neurodevelopmental Disorders * genetics physiopathology metabolism MeSH
- Receptors, N-Methyl-D-Aspartate * genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
N-methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimer's disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity "membrane-to-channel inhibition" (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg2+. K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30 μM K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by ∼51 %, outperforming 30 μM memantine (∼21 % inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15 mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10 mg/kg dosage resulted in brain concentrations of approximately 2 μM, with peak concentrations (Tmax) achieved within 15 minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060's potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.
- MeSH
- Excitatory Amino Acid Antagonists pharmacology MeSH
- Dizocilpine Maleate * pharmacology MeSH
- Excitatory Postsynaptic Potentials drug effects MeSH
- Hippocampus drug effects metabolism MeSH
- Humans MeSH
- Memantine pharmacology MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Neurons drug effects metabolism MeSH
- Receptors, N-Methyl-D-Aspartate * antagonists & inhibitors metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Opioid analgesics remain widely used for pain treatment despite the related serious side effects. Some of those, such as opioid tolerance and opioid-induced hyperalgesia may be at least partially due to modulation of opioid receptors (OR) function at nociceptive synapses in the spinal cord dorsal horn. It was suggested that increased release of different chemokines under pathological conditions may play a role in this process. The goal of this study was to investigate the crosstalk between the μOR, transient receptor potential vanilloid 1 (TRPV1) receptor and C-C motif ligand 2 (CCL2) chemokine and the involvement of spinal microglia in the modulation of opioid analgesia. METHODS: Patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) and dorsal root evoked currents (eEPSC) in spinal cord slices superficial dorsal horn neurons were used to evaluate the effect of μOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), CCL2, TRPV1 antagonist SB366791 and minocycline. Paw withdrawal test to thermal stimuli was combined with intrathecal (i.t.) delivery of CCL2 and DAMGO to investigate the modulation in vivo. RESULTS: Application of DAMGO induced a rapid decrease of mEPSC frequency and eEPSC amplitude, followed by a delayed increase of the eESPC amplitude, which was prevented by SB366791. Chemokine CCL2 treatment significantly diminished all the DAMGO-induced changes. Minocycline treatment prevented the CCL2 effects on the DAMGO-induced eEPSC depression, while mEPSC changes were unaffected. In behavioral experiments, i.t. injection of CCL2 completely blocked DAMGO-induced thermal hypoalgesia and intraperitoneal pre-treatment with minocycline prevented the CCL2 effect. CONCLUSIONS: Our results indicate that opioid-induced inhibition of the excitatory synaptic transmission could be severely attenuated by increased CCL2 levels most likely through a microglia activation-dependent mechanism. Delayed potentiation of neurotransmission after μOR activation is dependent on TRPV1 receptors activation. Targeting CCL2 and its receptors and TRPV1 receptors in combination with opioid therapy could significantly improve the analgesic properties of opioids, especially during pathological states.
- MeSH
- Anilides pharmacology MeSH
- Chemokine CCL2 pharmacology MeSH
- Cinnamates pharmacology MeSH
- Enkephalin, Ala(2)-MePhe(4)-Gly(5)- pharmacology MeSH
- Excitatory Postsynaptic Potentials drug effects MeSH
- Rats MeSH
- Spinal Cord drug effects MeSH
- Miniature Postsynaptic Potentials drug effects MeSH
- Synaptic Transmission drug effects MeSH
- Neurons drug effects MeSH
- Nociception drug effects MeSH
- Analgesics, Opioid pharmacology MeSH
- Rats, Wistar MeSH
- Spinal Cord Dorsal Horn drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The mechanisms of inflammatory pain need to be identified in order to find new superior treatments. Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) are highly co-expressed in dorsal root ganglion neurons and implicated in pain development. Here, we examined the role of spinal PAR2 in hyperalgesia and the modulation of synaptic transmission in carrageenan-induced peripheral inflammation, using intrathecal (i.t.) treatment in the behavioral experiments and recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs and eEPSCs) in spinal cord slices. Intrathecal PAR2-activating peptide (AP) administration aggravated the carrageenan-induced thermal hyperalgesia, and this was prevented by a TRPV1 antagonist (SB 366791) and staurosporine i.t. pretreatment. Additionally, the frequency of the mEPSC and sEPSC and the amplitude of the eEPSC recorded from the superficial dorsal horn neurons were enhanced after acute PAR2 AP application, while prevented with SB 366791 or staurosporine pretreatment. PAR2 antagonist application reduced the thermal hyperalgesia and decreased the frequency of mEPSC and sEPSC and the amplitude of eEPSC. Our findings highlight the contribution of spinal PAR2 activation to carrageenan-induced hyperalgesia and the importance of dorsal horn PAR2 and TRPV1 receptor interactions in the modulation of nociceptive synaptic transmission.
- MeSH
- Anilides pharmacology MeSH
- Posterior Horn Cells drug effects metabolism physiology MeSH
- Cinnamates pharmacology MeSH
- Excitatory Postsynaptic Potentials MeSH
- Hyperalgesia etiology metabolism physiopathology MeSH
- Carrageenan pharmacology toxicity MeSH
- TRPV Cation Channels antagonists & inhibitors metabolism MeSH
- Rats MeSH
- Miniature Postsynaptic Potentials MeSH
- Nociception MeSH
- Rats, Wistar MeSH
- Receptor, PAR-2 metabolism MeSH
- Staurosporine pharmacology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cholesterol is a structural component of cellular membranes particularly enriched in synapses but its role in synaptic transmission remains poorly understood. We used rat hippocampal cultures and their acute cholesterol depletion by methyl-β-cyclodextrin as a tool to describe the physiological role of cholesterol in glutamatergic synaptic transmission. Cholesterol proved to be a key molecule for the function of synapses as its depletion resulted in a significant reduction of both NMDA receptor (NMDAR) and AMPA/kainate receptor-mediated evoked excitatory postsynaptic currents (eEPSCs), by 94% and 72%, respectively. We identified two presynaptic and two postsynaptic steps of synaptic transmission which are modulated by cholesterol and explain together the above-mentioned reduction of eEPSCs. In the postsynapse, we show that physiological levels of cholesterol are important for maintaining the normal probability of opening of NMDARs and for keeping NMDARs localized in synapses. In the presynapse, our results favour the hypothesis of a role of cholesterol in the propagation of axonal action potentials. Finally, cholesterol is a negative modulator of spontaneous presynaptic glutamate release. Our study identifies cholesterol as an important endogenous regulator of synaptic transmission and provides insight into molecular mechanisms underlying the neurological manifestation of diseases associated with impaired cholesterol synthesis or decomposition.
- MeSH
- Receptors, AMPA metabolism MeSH
- Cholesterol pharmacology MeSH
- Excitatory Postsynaptic Potentials drug effects MeSH
- Hippocampus drug effects metabolism MeSH
- Rats MeSH
- Glutamic Acid metabolism MeSH
- Cerebral Cortex drug effects metabolism MeSH
- Synaptic Transmission * MeSH
- Neurons drug effects metabolism MeSH
- Rats, Wistar MeSH
- Presynaptic Terminals drug effects metabolism MeSH
- Receptors, N-Methyl-D-Aspartate metabolism MeSH
- Synapses drug effects metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Background: Depth and orientation of concentric needle electrode (CNE) insertion during the needle electromyography (EMG) might have influences on motor unit potentials (MUPs). Objective: To examine whether the depth of CNE insertion in m.vastus lateralis significantly affects PMU parameters in patients with myopathy (muscular dystrophy) and neuropathy (hereditary motor and sensory neuropathy). Methods: In order to analyze influences of depth and orientation of insertion of CNE, 11 patients with different neuromuscular disorders (8 with myopathies, and 3 with neuropathies) were examined. MUAPs parameters were compared after automatic (depths of perpendicular CNE insertion were 0, 0.5, 1, 2 and 3 cm in MVL) and manual method of extraction (0 and 1.5 cm). Results: MVL MUAP analysis (with typically changed MUPs features, especially duration) in patients with different neuromuscular disorders (neuropathies, myopathies) showed similar results like in healthy volunteers. Automated analysis of MVL in patients with myopathies showed irregular changes of the most of MUAPs parameters, especially duration. With depth of needle insertion, duration of MUAPs recorded with CNE and it’s componentes is not changed signifficantly, but that was not case with amplitude and area. Conclusion: A discrepancy between MUAPs parameters detected with manual and automatic methods is confirmed. We think that it is necessary to use specific standards for patients with neuropathic and myopathic diseases.
In this paper we investigate the rate coding capabilities of neurons whose input signal are alterations of the base state of balanced inhibitory and excitatory synaptic currents. We consider different regimes of excitation-inhibition relationship and an established conductance-based leaky integrator model with adaptive threshold and parameter sets recreating biologically relevant spiking regimes. We find that given mean post-synaptic firing rate, counter-intuitively, increased ratio of inhibition to excitation generally leads to higher signal to noise ratio (SNR). On the other hand, the inhibitory input significantly reduces the dynamic coding range of the neuron. We quantify the joint effect of SNR and dynamic coding range by computing the metabolic efficiency-the maximal amount of information per one ATP molecule expended (in bits/ATP). Moreover, by calculating the metabolic efficiency we are able to predict the shapes of the post-synaptic firing rate histograms that may be tested on experimental data. Likewise, optimal stimulus input distributions are predicted, however, we show that the optimum can essentially be reached with a broad range of input distributions. Finally, we examine which parameters of the used neuronal model are the most important for the metabolically efficient information transfer.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Action Potentials physiology MeSH
- Excitatory Postsynaptic Potentials physiology MeSH
- Membrane Potentials physiology MeSH
- Models, Neurological * MeSH
- Neural Conduction physiology MeSH
- Synaptic Transmission physiology MeSH
- Neural Inhibition physiology MeSH
- Neurons physiology MeSH
- Computer Simulation MeSH
- Signal-To-Noise Ratio MeSH
- Computational Biology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Paclitaxel chemotherapy treatment often leads to neuropathic pain resistant to available analgesic treatments. Recently spinal Toll-like receptor 4 (TLR4) and the transient receptor potential cation channel subfamily V member 1 (TRPV1) were identified to be involved in the pro-nociceptive effect of paclitaxel. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinases in this process, with the use of their antagonists (wortmannin, LY-294002, and staurosporine). The single paclitaxel administration (8 mg/kg i.p.) in mice induced robust mechanical allodynia measured as a reduced threshold to von Frey filament stimulation and generated reduced tachyphylaxis of capsaicin-evoked responses, recorded as changes in mEPSC frequency in patch-clamp recordings of dorsal horn neurons activity in vitro, for up to eight days. Paclitaxel application also induced increased Akt kinase phosphorylation in rat DRG neurons. All these paclitaxel-induced changes were prevented by the wortmannin in vivo pretreatment. Acute co-application of wortmannin or LY-294002 with paclitaxel in spinal cord slices also attenuated the paclitaxel effect on capsaicin-evoked responses. Staurosporine was effective in the acute in vitro experiments and on the first day after the paclitaxel treatment in vivo, but in contrast to wortmannin, it did not have a significant impact later. Our data suggest that the inhibition of PI3K signaling may help alleviate pathological pain syndromes in the paclitaxel-induced neuropathy.
- MeSH
- Posterior Horn Cells drug effects metabolism MeSH
- Excitatory Postsynaptic Potentials drug effects MeSH
- Phosphatidylinositol 3-Kinases metabolism MeSH
- Hyperalgesia chemically induced drug therapy metabolism MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- Capsaicin pharmacology MeSH
- Transient Receptor Potential Channels MeSH
- TRPV Cation Channels metabolism MeSH
- Rats MeSH
- Lipopolysaccharides pharmacology MeSH
- Spinal Cord drug effects metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Neuralgia chemically induced drug therapy metabolism MeSH
- Oncogene Protein v-akt metabolism MeSH
- Paclitaxel toxicity MeSH
- Peptide Fragments immunology metabolism MeSH
- Rats, Wistar MeSH
- Protein Serine-Threonine Kinases antagonists & inhibitors metabolism MeSH
- Protein Kinase C immunology metabolism MeSH
- Signal Transduction drug effects MeSH
- Toll-Like Receptor 4 metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Inhibitory circuits in the auditory brainstem undergo multiple postnatal changes that are both activity-dependent and activity-independent. We tested to see if the shift from GABA- to glycinergic transmission, which occurs in the rat medial nucleus of the trapezoid body (MNTB) around the onset of hearing, depends on sound-evoked neuronal activity. We prevented the activity by bilateral cochlear ablations in early postnatal rats and studied ionotropic GABA and glycine receptors in MNTB neurons after hearing onset. The removal of the cochlea decreased responses of GABAA and glycine receptors to exogenous agonists as well as the amplitudes of inhibitory postsynaptic currents. The reduction was accompanied by a decrease in the number of glycine receptor- or vesicular GABA transporter-immunopositive puncta. Furthermore, the ablations markedly affected the switch in presynaptic GABAA to glycine receptors. The increase in the expression of postsynaptic glycine receptors and the shift in inhibitory transmitters were not prevented. The results suggest that inhibitory transmission in the MNTB is subject to multiple developmental signals and support the idea that auditory experience plays a role in the maturation of the brainstem glycinergic circuits.
- MeSH
- Ablation Techniques * MeSH
- GABA-A Receptor Agonists pharmacology MeSH
- Trapezoid Body physiology MeSH
- Inhibitory Postsynaptic Potentials physiology MeSH
- Cochlea physiopathology surgery MeSH
- Rats MeSH
- Synaptic Transmission * MeSH
- Neural Inhibition drug effects physiology MeSH
- Animals, Newborn MeSH
- Receptors, GABA-A physiology MeSH
- Receptors, Glycine agonists metabolism physiology MeSH
- Evoked Potentials, Auditory, Brain Stem physiology MeSH
- Vesicular Inhibitory Amino Acid Transport Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
